Heather R. Ferguson Bennit

Early diagnostic value of survivin and its alternative splice variants in breast cancer.

BackgroundThe inhibitor of apoptosis (IAP) protein Survivin and its splice variants are differentially expressed in breast cancer tissues. Our previous work showed Survivin is released from tumor cells via small membrane-bound vesicles called exosomes. We, therefore, hypothesize that analysis of serum exosomal Survivin and its splice variants may provide a novel biomarker for early diagnosis of breast cancer.MethodsWe collected sera from forty breast cancer patients and ten control patients who were disease free for 5 years after treatment. In addition, twenty-three paired breast cancer tumor tissues from those same 40 patients were analyzed for splice variants. Serum levels of Survivin were analyzed using ELISA and exosomes were isolated from this serum using the commercially available ExoQuick kit, with subsequent Western blots and immunohistochemistry performed.ResultsSurvivin levels were significantly higher in all the breast cancer samples compared to controls (p < 0.05) with exosome amounts significantly higher in cancer patient sera compared to controls (p < 0.01). While Survivin and Survivin-∆Ex3 splice variant expression and localization was identical in serum exosomes, differential expression of Survivin-2B protein existed in the exosomes. Similarly, Survivin and Survivin-∆Ex3 proteins were the predominant forms detected in all of the breast cancer tissues evaluated in this study, whereas a more variable expression of Survivin-2B level was found at different cancer stages.ConclusionIn this study we show for the first time that like Survivin, the Survivin splice variants are also exosomally packaged in the breast cancer patients’ sera, mimicking the survivin splice variant pattern that we also report in breast cancer tissues. Differential expression of exosomal-Survivin, particularly Survivin-2B, may serve as a diagnostic and/or prognostic marker, a “liquid biopsy” if you will, in early breast cancer patients. Furthermore, a more thorough understanding of the role of this prominent antiapoptotic pathway could lead to the development of potential therapeutics for breast cancer patients.

Curcumin Modulates Pancreatic Adenocarcinoma Cell-Derived Exosomal Function

Pancreatic cancer has the highest mortality rates of all cancer types. One potential explanation for the aggressiveness of this disease is that cancer cells have been found to communicate with one another using membrane-bound vesicles known as exosomes. These exosomes carry pro-survival molecules and increase the proliferation, survival, and metastatic potential of recipient cells, suggesting that tumor-derived exosomes are powerful drivers of tumor progression. Thus, to successfully address and eradicate pancreatic cancer, it is imperative to develop therapeutic strategies that neutralize cancer cells and exosomes simultaneously. Curcumin, a turmeric root derivative, has been shown to have potent anti-cancer and anti-inflammatory effects in vitro and in vivo. Recent studies have suggested that exosomal curcumin exerts anti-inflammatory properties on recipient cells. However, curcumin’s effects on exosomal pro-tumor function have yet to be determined. We hypothesize that curcumin will alter the pro-survival role of exosomes from pancreatic cancer cells toward a pro-death role, resulting in reduced cell viability of recipient pancreatic cancer cells. The main objective of this study was to determine the functional alterations of exosomes released by pancreatic cancer cells exposed to curcumin compared to exosomes from untreated pancreatic cancer cells. We demonstrate, using an in vitro cell culture model involving pancreatic adenocarcinoma cell lines PANC-1 and MIA PaCa-2, that curcumin is incorporated into exosomes isolated from curcumin-treated pancreatic cancer cells as observed by spectral studies and fluorescence microscopy. Furthermore, curcumin is delivered to recipient pancreatic cancer cells via exosomes, promoting cytotoxicity as demonstrated by Hoffman modulation contrast microscopy as well as AlamarBlue and Trypan blue exclusion assays. Collectively, these data suggest that the efficacy of curcumin may be enhanced in pancreatic cancer cells through exosomal facilitation.

The emerging role of exosomes in survivin secretion.

The tumor microenvironment plays an integral part in the biology of cancer, participating in tumor initiation, progression, and response to therapy. Factors released by tumor cells themselves contribute in creating an environment mostly favorable but sometimes detrimental to the tumor. Survivin, one of the key members of the inhibitor of apoptosis (IAP) family of proteins, has been shown in the cytoplasm, mitochondria, nucleus, and most recently in the extracellular space, transported via small membrane bound vesicles called exosomes. Exosomes are secreted from hematopoietic, non-hematopoietic, tumor, and non-tumor cells, shuttling essential molecules such as proteins, RNAs, and microRNAs, all believed to be important for cell-cell and cell-extracellular communication. In this review, we discuss exosomal Survivin and its role in modifying the tumor microenvironment.

Exosomes Secreted from Human Cancer Cell Lines Contain Inhibitors of Apoptosis (IAP)

Exosomes are endosomal-derived nanovesicles released by normal and tumor cells which have been shown to transfer functionally active protein, lipids, mRNAs and miRNAs between cells. Varying in molecular profiles, biological roles, functional roles and protein contents, exosomes have been described as “multi-purpose carriers” playing a role in supporting the survival and growth of tumor cells. The IAP Survivin has been found to be present in tumor exosomes. However, the existence of other IAPs in tumor exosomes is still unknown. Survivin, cIAP1, cIAP2 and XIAP mRNA and protein are differently expressed in a panel of tumor cell lines: DLCL2, HeLa, MCF-7, Panc-1, and PC3. Exosomes were isolated from conditioned media collected from the cells from which RNA and protein were extracted. Our results provide evidence that like Survivin, XIAP, cIAP1 and cIAP2 proteins are found in tumor exosomes. The mRNA expression, however, is differentially expressed across the tumor cell lines. The presence of these bioactive molecules in exosomes may not only serve as warning signals, but also play a role in providing protection to the cancer cells against changes that are constantly occurring in the tumor microenvironment.

Abstract 1108: B-cell lymphoma-derived exosomes are reservoirs of inhibitors of apoptosis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA B-cells, along with other normal and cancerous cells, secrete 50-150 nm vesicles called exosomes. Exosomes carry proteins, mRNA, and miRNA that can be functional upon cellular uptake and are often specific to cell of origin, offering potential as disease biomarkers. Our lab has identified within exosomes from solid tumor cells a cancer-specific protein, Survivin, which is a member of the Inhibitors of Apoptosis (IAP) family. Whether Survivin and other IAP family members such as XIAP, cIAP1 or cIAP2 are also expressed in exosomes from hematopoietic malignancies is yet to be determined. In this study, we aim to investigate the exosomes released from B-cell lymphoma cells and determine the IAP content under basal and sublethal stress conditions. Three human non EBV-transformed B-cell lymphoma lines developed at Wayne State University were used. WSU-FSCCL (follicular small cleaved cell), WSU-DLCL-2 (diffuse large cell), and WSU-WM (Waldenstrom Macroglobulinemia) represent different levels of aggressiveness. Cells were cultured for 24 or 48 hours before exosome isolation from the conditioned media using either ultracentrifugation on a sucrose cushion or ExoQuick (System Biosciences). Sublethal levels of UV irradiation were determined for each cell line by analyzing DNA content by propidium iodide staining with a FACSCalibur. Protein quantity of exosomes was measured using a BCA assay, and protein content was studied using Western blotting. Corresponding gene expression was identified using RT-PCR. Here we report IAP protein and mRNA (except XIAP) within exosomes from basal cells. We are currently probing exosomes from stressed cells for comparison. We hypothesize that the IAP exosomal profile may change under stressed conditions. The specificity and variability in exosomal content offers capabilities not only as disease biomarkers, but also in the immunomodulating activity of exosomes which have shown roles in both stimulating and inhibiting the immune system, We have found evidence that exosomal Survivin plays an immune role in skewing Th1/Th2 populations and decreasing cytotoxic function. Changes in exosome content between basal and stressed cells may have roles in the immunomodulation of the tumor microenvironment which facilitates the evasion of lymphoma cells to immune destruction, as well as possible prognostic benefit as to whether treatments are being effective or detrimental. Citation Format: Heather R. Ferguson Bennit, Malyn M. Asuncion Valenzuela, Jessica S. Jutzy, Nathan R. Wall. B-cell lymphoma-derived exosomes are reservoirs of inhibitors of apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1108. doi:10.1158/1538-7445.AM2014-1108

Uptake of lymphoma-derived exosomes by peripheral blood leukocytes

Exosomes are nanosized lipid vesicles secreted into blood and other body fluids and serve as vehicles for intercellular communication. Despite being an important component of the tumor microenvironment (TME), exosomal targeting and uptake into recipient cells are still not fully understood. Few studies have looked at lymphoma exosomes and their interactions with circulating blood cells. In this study, we examine the exosomal uptake distribution among peripheral blood leukocytes (PBLs) using vesicles derived from a diffuse large B cell lymphoma cell line, WSU-DLCL2. Lymphoma cells survive, proliferate, and are protected from the cytotoxic effects of chemotherapeutic agents by soluble factors or by direct contact with inflammatory and stromal cells within the TME. In an attempt to close the gap in knowledge concerning lymphoma TME immunosuppression, we have treated normal human PBLs with PKH67-labeled lymphoma exosomes and monitored the uptake by measuring fluorescence at different time points using flow cytometry and fluorescent microscopy. Our results show that of the four populations examined, B cells and monocytes demonstrated uptake of PKH67-labeled exosomes, while T cells and NK cells displayed significantly less uptake.

Exosomal survivin facilitates vesicle internalization

Survivin, a member of the inhibitor of apoptosis (IAP) protein family plays a significant role in cell fate and function. It is significantly overexpressed in tumor cells and has been identified in most cancer cell types. A novel extracellular population has recently been identified and its function is still unknown. Emerging evidence continues to shed light on the important role the tumor microenvironment (TME) has on tumor survival and progression. This new population of survivin has been seen to enhance the tumor phenotype when internalized by recipient cells. In this paper, we sought to better understand the mechanism by which survivin is taken up by cancer cells and the possible role it plays in this phenomenon. We isolated the exosomal carriers of extracellular survivin and using a lipophilic stain, PKH67, we tracked their uptake with immunofluorescence and flow cytometry. We found that by blocking exosomal survivin, exosome internalization is reduced, signifying a novel function for this protein. We also discovered that the common membrane receptors, transferrin receptor, endothelin B receptor, insulin receptor alpha, and membrane glucocorticoid receptor all facilitate exosomal internalization. This understanding further clarifies the protein-protein interactions in the TME that may influence tumor progression and identifies additional potential chemotherapeutic targets.

Peripheral Blood Cell Interactions of Cancer-Derived Exosomes Affect Immune Function

Cancer-derived exosomes are constitutively produced and secreted into the blood and biofluids of their host patients providing a liquid biopsy for early detection and diagnosis. Given their ubiquitous nature, cancer exosomes influence biological mechanisms that are beneficial to the tumor cells where they are produced and the microenvironment in which these tumors exist. Accumulating evidence suggests that exosomes transport proteins, lipids, DNA, mRNA, miRNA and long non coding RNA (lncRNA) for the purpose of cell-cell and cell-extracellular communication. These exosomes consistently reflect the status as well as identity of their cell of origin and as such may conceivably be affecting the ability of a functional immune system to recognize and eliminate cancer cells. Recognizing and mapping the pathways in which immune suppression is garnered through these tumor derived exosome (TEX) may lead to treatment strategies in which specific cell membrane proteins or receptors may be targeted, allowing for immune surveillance to once again help with the treatment of cancer. This Review focuses on how cancer exosomes interact with immune cells in the blood.

Abstract A03: Effects of survivin and lymphoma cell-derived exosomes on natural killer cell function

Tumors alter their microenvironment to promote survival using methods such as angiogenesis promotion, growth signals, and immune suppression. The immune system, whose regular function is to recognize and eradicate infected or abnormal cells, often seems unresponsive to transformed neoplastic cells. Many of the methods employed by tumors to reduce immune function are unclear, but there is evidence for T cell suppression, increased myeloid-derived suppressor cells (MDSCs), and reduced natural killer (NK) cell activity. NK cells are among the first cells to recognize and destroy abnormal cells due to their inherent killing capabilities. However, many types of cancers inhibit the surveillance and cytotoxic abilities of NK cells by releasing exosomes, vesicles that can modulate tumor microenvironment and intercellular communication for the purpose of enhancing tumor malignancy. Exosomes as a means for immunomodulation of tumor microenvironment have been the focus of recent research. These 50-150 nm sized lipid bound vesicles are secreted by many cell types, including immune cells and tumor cells, and the unique protein, lipid, mRNA and miRNA contents contribute to the complex intercellular communication occurring between malignant and normal cells. Cancer patients often have increased numbers of exosomes circulating through their body, including patients with hematological malignancies, such as lymphoma. Recently, cancer cell exosomes have been found to contain Survivin, an Inhibitor of Apoptosis (IAP) protein that prevents cell death and induces Th2 polarity shift and decreases proliferation and cytotoxicity of CD8+ T lymphocytes. The purpose of this study is to explore the effect of Survivin and cancer cell-derived exosomes, which contain Survivin, on the immune functions of NK cells. The hypothesis is that Survivin and exosomes will decrease NK cell functions by suppressing NK cell production or release of cytokines and cytotoxic granules. NK cells were obtained from peripheral blood using magnetic NK cell isolation kit from Miltenyi Biotec and exosomes were obtained from conditioned media from two lymphoma cell lines (WSU-DLCL2 and WSU-FSCCL) using ExoQuickTM (System Biosciences). Flow cytometry methods were used to evaluate degranulation capacity by measuring levels of CD107a, as well as expression of activating receptor NKG2D, cytotoxic granules (perforin, Granzyme B) and cytokines (TNF-α, IFN-γ). RNA message expression was investigated using both block RT-PCR and qPCR. Results showed little significant difference in ability to degranulate when exposed to stimuli, and NKG2D levels did not change after exposure to Survivin or lymphoma exosomes. Although ImageJ analysis of block PCR showed some instances of decreased band densities in some donors treated with Survivin protein, RNA levels in general did not show a trend between NK donors in response to treatment. However, protein expression of Granzyme B, perforin, TNF-α, and IFN-γ did appear to decrease after Survivin treatment. This work is still in the early stages and results are inconsistent due to donor variability. More samples are required, as well as further studies on NK cell functional cytotoxicity. Citation Format: Heather R. Ferguson Bennit, Amber Gonda, Jenniffer Licero Campbell, Laura Oppegard, David Chi, Nathan R. Wall. Effects of survivin and lymphoma cell-derived exosomes on natural killer cell function. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A03.

Abstract 5089: Extracellular Survivin uptake by cancer cells via receptor-mediated endocytosis

The tumor microenvironment is a crucial player in tumorigenesis, proliferation, and survival. Communication between its various components through microvesicular trafficking is central to the interaction between tumor cells and the surrounding milieu. Exosomes, nanovesicles released by all cell types, carry various proteins and nucleic acids. Once released into the extracellular environment, exosomes are taken up by surrounding cells or the exosomes travel in various bodily fluids to reach distant parts of the body. Those exosomes derived from tumor cells contain proteins and nucleic acids essential to the development and maintenance of tumors. One such protein, Survivin, a member of the Inhibitor of Apoptosis (IAP) protein family, has been identified in virtually all cancer cells. Survivin function is closely linked with subcellular location. Nuclear Survivin plays a key role in cell division and cell cycle regulation as a member of the chromosome passenger complex. Cytosolic and mitochondrial Survivin primarily facilitate the inhibition of apoptosis. Recent research has identified a unique pool of Survivin in the extracellular environment. Functions tied to this location are as yet undefined, however, evidence suggests that this population may contribute to increased proliferation, invasiveness and therapeutic resistance when taken into recipient cancer cells. Using tandem affinity purification we have identified a relationship between Survivin and TNF receptor 1 (TNFR1), as well as transferrin receptor 2 (TfR2). Both of these receptors play a role in endocytosis of extracellular proteins. Transferrin receptors are upregulated on cancer cells to facilitate the increased iron demand of proliferating cells. We therefore hypothesize that exosomal Survivin is taken up by adjacent cancer cells via receptor-mediated endocytosis. We have shown that in the presence of antibodies to these receptors, there is a decreased uptake of extracellular Survivin (from conditioned media). Results were similar when conditioned media was introduced to HeLa cells with siRNA knockdowns of each receptor, as well as to CHO cells deficient in these receptors. We conclude that uptake of extracellular Survivin into cancer cells is dependent upon these two endocytosis receptors. The relationship of Survivin and exosomes in the extracellular environment is indicative that exosomes transport Survivin out of tumor cells and into the surroundings. These results suggest that exosomal Survivin enters cancer cells by way of receptor-mediated endocytosis. Understanding this mechanism of signaling between cancer cells shows the potential intercellular trafficking of oncoproteins may play in tumor maintenance and progression. It also provides us with a novel means to combat the resulting enhanced aggressive nature of the disease. Citation Format: Amber Gonda, Salma Khan, Heather R. Ferguson Bennit, Ron B. Moyron, Nathan R. Wall. Extracellular Survivin uptake by cancer cells via receptor-mediated endocytosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5089.